Quercetin-induced upregulation of human GCLC gene is mediated by cis-regulatory element for early growth response protein-1 (EGR1) in INS-1 beta-cells.

The catalytic subunit of gamma-glutamylcysteine ligase (GCLC) catalyses the rate-limiting step in the de novo synthesis of glutathione (GSH), which is involved in maintaining intracellular redox balance. GSH is especially important for antioxidant defense system since beta-cells show intrinsically low expression of antioxidant enzymes. In the present study, we investigated the regulatory mechanisms by which quercetin, a flavonoid, induces the expression of the GCLC gene in rat pancreatic beta-cell line INS-1. Promoter study found that the proximal GC-rich region (from -90 to -34) of the GCLC promoter contained the quercetin-responsive cis-element(s). The quercetin-responsive region contains consensus DNA binding site for early growth response 1 (EGR1) at -67 (5'-CGCCTCCGC-3') which overlaps with a putative Sp1 binding site. Electrophoretic mobility shift assay showed that an oligonucleotide containing the EGR1 site was bound to nuclear factors EGR1, Sp1, and Sp3. In the promoter analysis, mutation of EGR1 site significantly reduced the quercetin response, whereas mutation of Sp1 site decreased only the basal activity of the GCLC promoter. Additionally, the transient overexpression of EGR1 significantly increased basal activity of the GCLC promoter. Finally, we showed that quercetin potently induced both EGR1 mRNA and its protein levels without affecting the expression of Sp1 and Sp3 proteins. Therefore, we concluded that EGR1 was bound to GC-rich region of the GCLC gene promoter, which was prerequisite for the transactivation of the GCLC gene by quercetin.